Phage therapy of Pseudomonas infections

ABSTRACT

The present invention relates to bacteriophage therapy. More particularly, the present invention relates to novel bacteriophages having a high specificity against  Pseudomonas aeruginosa  strains, their manufacture, components thereof, compositions comprising the same and the uses thereof in phage therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/031,763, filed Apr. 25, 2016, now U.S. Pat. No. 10,077,431, which isthe U.S. national stage application of International Patent ApplicationNo. PCT/EP2014/072905, filed Oct. 24, 2014.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Apr. 8, 2016 and is 934 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

The present invention relates to novel bacteriophages, compositionscomprising the same, their manufacture, and the uses thereof. Theinvention is particularly adapted for the treatment of an infection in amammal and for improving a subject condition by modifying the flora insaid subject.

BACKGROUND OF THE INVENTION

Bacteriophages (or phages) are small viruses displaying the ability toinfect and kill bacteria while they do not affect cells from otherorganisms. Initially described almost a century ago by William Twort,and independently discovered shortly thereafter by Félix d'Herelle, morethan 6000 different bacteriophages have been discovered so far anddescribed morphologically, including bacterial and archaeal viruses. Thevast majority of these viruses are tailed while a small proportion arepolyhedral, filamentous or pleomorphic. They may be classified accordingto their morphology, their genetic content (DNA vs. RNA), their specifichost, the place where they live (marine virus vs. other habitats), andtheir life cycle. As intra-cellular parasites of bacterial cells, phagesdisplay different life cycles within the bacterial host: lytic,lysogenic, pseudo-lysogenic, and chronic infection (Weinbauer, 2004;Drulis-Kawa, 2012). Lytic phages cause lysis of the host bacterial cellas a normal part of their life cycle. Lysogenic phages (also termedtemperate phages) can either replicate by means of the lytic life cycleand cause lysis of the host bacterium, or they can incorporate their DNAinto the host bacterial DNA and become noninfectious prophages. Whateverthe type of cycle of a phage life, the first step is the attachment toreceptors of the bacterial cell wall before phage material may enter thebacteria. This specific process influences the spectrum of the possiblephage-bacteria interactions.

Bacteriophages are commonly used as research tools to modify bacteria inlaboratory experiments.

Because of their target host cell specificity, the use of phages as atherapy to treat acute and chronic infections has been considered,particularly in dermatology, ophthalmology, urology, stomatology,pediatrics, otolaryngology or surgery. This concept of therapeutic useof phages to treat bacterial infection was, however, highlycontroversial from the very beginning and not widely accepted by thepublic or medical community. Early studies were widely criticized forlack of appropriate controls and inconsistent results. The lack ofreproducibility and many conflicting results obtained in the variouspublished studies led the Council on Pharmacy and Chemistry of theAmerican Medical Association to conclude that the evidence for thetherapeutic value of lytic filtrates was for the most part contradictoryand unconvincing, and recommend additional research to confirm itspurported benefits.

Since the introduction of antibiotics in the 1940s, little attention waspaid to this field of therapeutics, especially in the Western world. Butthe extensive use of antibiotics has led to the widespread emergence andspread of antibiotic-resistant bacteria around the world, causingincreasingly serious problems. It has therefore become a majortherapeutic challenge to overcome the limited therapeutic optionsremaining to treat major multi-drug resistant microbes.

In addition, many pathogenic microorganisms reside within biofilms,which biofilms cause additional problems when designing newanti-microbial agents. In this regard, bacteria growing as a biofilmrather than in single-celled (“planktonic”) forms tend to beparticularly resistant to anti-microbial agents and to be particularlydifficult for the host immune system to render an appropriate response.

Since its initial discovery in the late 19th century (Fordos 1859), theGram-negative bacterium Pseudomonas aeruginosa has gained a notoriousplace in the list of infamous human pathogens (Williams et al., 1894;Freeman et al., 1916). The arrival of the antibiotic era largelypalliated the previously fatal outcome of acute infections in healthypatients. Only a relative improvement has been achieved in theeradication of chronic infections, which develop mainly in individualssuffering from cystic fibrosis or severe burns or who areimmunocompromised (Gang et al., 1999; Jones et al., 2010). Twointrinsically related factors in the fatal outcome of infection in thesepatients are the rapid prescription of inappropriate antibiotictreatments and the development or acquisition of multidrug-resistantstrains. While the use of (an) appropriate antibiotic(s) has beenreported as an essential factor in the eradication of P. aeruginosainfections (Kang et al., 2005; Micek et al., 2005), conversely,antibiotic abuse significantly contributes to increasing resistance byexerting a continuous selective pressure for the acquisition of suchcapabilities. Antibiotics alone do not account for the high prevalenceof multidrug-resistant variants: P. aeruginosa has multiple,chromosomally encoded intrinsic mechanisms of resistance, including lowpermeability of the cell envelope and numerous multidrug efflux pumps.Another major factor accounting for the successful invasive behavior andpersistence of this bacterium is its high adaptability, allowing rapidcolonization of different environments.

Furthermore, pathogenic bacteria such as P. aeruginosa are able to formbiofilms, which contribute to their increased resistance to antibiotics.Such biofilms may comprise more than one type of bacteria supported andsurrounded by an excreted extracellular matrix, and assist bacteria tocolonize various surfaces. Biofilms allow bacteria to attach to surfacesand to reach population densities which would otherwise beunsupportable, imparting increased resistance to not only antibioticsbut many environmental stresses including toxins such as heavy metals,bleaches and other cleaning agents. It is known that bacteria withinbiofilms can be 100 to 1000 times more resistant to antibiotics than thesame strain of bacteria growing in planktonic forms. Such an increasedresistance means that bacteria that are apparently sensitive toantibiotics in a laboratory test may be resistant to therapy in aclinical setting. Even if some are cleared, biofilms may provideresistant reservoirs permitting rapid colonization once antibiotics areno longer present. It is therefore obvious that biofilms are majorfactors in many human diseases. Chemical treatments are unsuited for useagainst biofilms since this is precisely what they have evolved tocounter. Physical abrasion does provide a means to disrupt biofilms.Unfortunately, many surfaces where biofilms support bacterialpathogenesis are poorly suited to rigorous abrasion, i.e., bones,joints, implanted medical devices, etc. For example, the surfaces ofwounds or burns are extremely sensitive and delicate. Even whereabrasion is both suitable and in routine use, clearing of biofilms islimited. Oral plaque on the surface of teeth is a biofilm and ispartially cleared by regular brushing. However, bacteria are maintainedon unbrushed surfaces (for example in the gaps between teeth) and canrecolonize cleared surfaces both rapidly and effectively. From this, itis clear that existing approaches to clearing biofilms are of limitedefficacy.

The capability for quick adaptation and the ability to form biofilms arethe main reasons that identify P. aeruginosa as opportunistic pathogens.They have acquired the status of hospital pathogens, and may be isolatedfrom clinical samples taken from wounds, sputum, bladder, urethra,vagina, ears, eyes and respiratory tract. The emergence of resistance tothe most powerful new antibiotics in such clinical P. aeruginosastrains, occurring even during treatment, makes the fight with P.aeruginosa hospital pathogens a great problem.

Furthermore, it has been reported that the pathological or physiologicalcondition of a subject is influenced by the balance of microorganisms inthe flora of the subject. Accordingly, modifying the microbial flora, ormodifying said balance, or restoring said balance, by destroying P.aeruginosa population, is also a valuable approach for improving asubject condition.

Therefore, there is a great need for new antibacterial agents orcompositions that can be used to destroy P. aeruginosa strains, evenwhen organized in bacterial biofilms, suitable for use in human oranimal therapy, as well, as for decontaminating materials.

SUMMARY OF THE INVENTION

The inventors have isolated and characterized new bacteriophagespresenting specific lytic activity to Pseudomonas aeruginosa (P.aeruginosa), which can be used as active agents in pharmaceutical orveterinary preparations, particularly to treat P. aeruginosa bacterialinfections or to modify microbial balance in a subject. The newbacteriophages of the invention exhibit strong lytic activity and highselectivity, and can be combined to induce controlled destruction of avery large spectrum of P. aeruginosa cells.

An object of the invention is to provide antibacterial compositionscomprising at least one, preferably at least two bacteriophages havinglytic activity against a Pseudomonas aeruginosa (P. aeruginosa) strain,said bacteriophages being selected from the bacteriophages having agenome comprising a nucleotide sequence of any one of SEQ ID NOs: 1 to13 or a sequence having at least 90% identity thereto.

A further object of the invention relates to a bacteriophage havinglytic activity to a Pseudomonas aeruginosa (P. aeruginosa) strain andhaving a genome comprising a nucleotide sequence selected from any oneof SEQ ID NOs: 1 to 13 or a sequence having at least 97% identitythereto.

The bacteriophages of the invention exhibit lytic activity to multi drugresistant strains of P. aeruginosa, in particular to antibioticresistant pathogenic strains, such as cephalosporinase,carbenicillinases and extended-spectrum β-lactamases (Strateva T. andYordanov D. 2009).

In another aspect, the invention is related to a bacteriophage havinglytic activity to a pathogenic P. aeruginosa strain, wherein thebacteriophage is specific for P. aeruginosa, active againstantibiotic-resistant P. aeruginosa strains, and has a productive lyticeffect below 20.

The invention further concerns an isolated nucleic acid contained in abacteriophage of the invention, preferably an isolated nucleic acidmolecule comprising a nucleotide sequence selected from any one of SEQID NOs: 1 to 13 or a sequence having at least 97% identity thereto, aswell as an isolated polypeptide encoded by said nucleic acid.

Another object of the invention is a composition comprising a nucleicacid or polypeptide as defined above.

The compositions of the invention typically further comprise apharmaceutically or veterinary acceptable excipient or carrier. They maybe liquid, semi-liquid, solid or lyophilized.

Another object of the invention relates to a bacteriophage, nucleicacid, polypeptide or composition as defined above, for use in thetreatment of an infection in a mammal, for modifying the microbial florain a mammal, for decontaminating a material and/or for killing a P.aeruginosa bacterium or for compromising the integrity of a bacterialbiofilm.

The invention relates also to the use of one or several lyticbacteriophages to improve a subject condition by modifying the microbialflora in said subject. The microbial flora may be modified bycorrecting, adapting or restoring a proper balance of microorganisms insaid flora.

The invention also relates to a method for treating an infection in amammal, comprising the administration to said mammal of at least onebacteriophage, nucleic acid, polypeptide or composition as definedabove.

The invention also relates to a method for treating a surface ormaterial suspected of being contaminated with a P. aeruginosa bacterium,comprising applying to said surface or material at least onebacteriophage, nucleic acid, polypeptide or composition as definedabove. The surface or material may be a surface of any device, vessel orlaboratory material, cloth, etc.

A further object of the invention relates to a kit comprising acomposition as defined above and a means for applying the same to asubject or surface.

Another object of the invention relates to a method for predicting ordetermining efficacy of a bacteriophage therapy in a subject, whereinthe method comprises determining in vitro a lytic activity of one ormore bacteriophages of the invention to a P. aeruginosa strain from asample of said subject, a lytic activity of one or more bacteriophagesof the invention to at least one P. aeruginosa strain from said samplebeing indicative of an efficient treatment. The method furtheroptionally comprises the step of treating the subject with at least onebacteriophage having a lytic activity to a P. aeruginosa strain from asample of said subject.

In another aspect, the invention provides a method for selecting asubject or determining whether a subject is susceptible to benefit froma bacteriophage therapy, wherein the method comprises the step ofdetermining in vitro a lytic activity of one or more bacteriophages ofthe invention to a P. aeruginosa strain from a sample of said subject, alytic activity of one or more of said bacteriophages to at least one P.aeruginosa strain being indicative of a responder subject.

The invention may be used in any mammal, preferably in human beings, orto treat any material, including laboratory materials or medicaldevices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: In vitro efficacy of Bacteriophages of the invention on variouscombinations of P. aeruginosa strains.

FIG. 2: In vivo efficacy of Bacteriophages of the invention on variouscombinations of P. aeruginosa strains.

FIG. 3: Efficacy of bacteriophages of the invention in vivo on Is580 P.aeruginosa strain-mediated infection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel bacteriophages, componentsthereof, compositions comprising the same, their manufacture, and theuses thereof as antibacterial agents, particularly for the treatment ofan infection in a mammal and for improving a subject condition bymodifying the microbial flora in said subject.

Definitions

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “bacteriophage” or “phage” refers to afunctional phage particle comprising a nucleic acid genome packaged in aproteinaceous envelope or capsid. The term also refers to portions ofthe bacteriophage, including, e.g., a head portion, or an assembly ofphage components, which provide substantially the same functionalactivity.

The term “phenotypic characteristic” designates more preferably themorphology and/or host-range of a bacteriophage. Methods for phenotypingbacteriophages are well known per se in the art and include, forexample, determining bacterial host range and/or activity against thebiofilm produced by certain bacterial strains.

The term “lytic activity” as used in the invention designates theproperty of a bacteriophage to cause lysis of a bacterial cell. Thelytic activity of a bacteriophage can be tested on P. aeruginosa strainsaccording to techniques known per se in the art (see also Examplessection).

The term “variant” of a reference bacteriophage designatesbacteriophages having variation(s) in the genomic sequence and/orpolypeptide(s) encoded thereby as compared to said referencebacteriophage, while retaining the same phenotypic characteristics asthe reference bacteriophage. Variants typically comprise, e.g., silentmutations, conservative mutations, minor deletions, and/or minorreplications of genetic material, and retains phenotypic characteristicsof the reference bacteriophage. In a preferred embodiment, the variantof the invention retain any observable characteristic or property thatis dependent upon the genome of the bacteriophage of the invention, i.e.phenotypic characteristics of said bacteriophage and/or lytic activityagainst the P. aeruginosa strains. Preferred variants have less than 5%nucleic acid variation as compared to the genome of the referencebacteriophage, even more preferably less than 4%, more preferably lessthan 2%. Alternatively, or in combination, variants have preferably lessthan 5% amino acid variation in a coded polypeptide sequence as comparedto a polypeptide of the reference bacteriophage.

The term “% identity” in relation to nucleic acid or amino acidsequences designates the level of identity or homology between saidsequences and may be determined by techniques known per se in the art.Typically, the % identity between two nucleic acid or amino acidsequences is determined by means of computer programs such as GAPprovided in the GCG program package (Program Manual for the WisconsinPackage, Version 8, August 1996, Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D.,1970, Journal of Molecular Biology, 48, 443-453). With settings adjustedto e.g., DNA sequences (particularly, GAP creation penalty of 5.0 andGAP extension penalty of 0.3), nucleic acid molecules may be aligned toeach other using the Pileup alignment software available as part of theGCG program package. The % identity between two sequences designates theidentity over the entire length of said sequences.

The term “fragment” of a nucleic acid designates typically a fragmenthaving at least 10 consecutive nucleotides of said nucleic acid, morepreferably at least 15, 20, 25, 30, 35, 40, 50 or more consecutivenucleotides of said nucleic acid.

The term “fragment” of a polypeptide designates typically a fragmenthaving at least 5 consecutive amino acids of said polypeptide, morepreferably at least 10, 15, 20, 30, 40, 50 or more consecutive aminoacids of said polypeptide.

The terms “ESBL P. aeruginosa strain” refers to cephalosporinase and/orextended-spectrum β-lactamases producing P. aeruginosa strains,including various forms of antibiotic resistance such as AmpCβ-lactamase or Class A carbenicillin hydrolyzing β-lactamases, etc.

The term “specific” or “specificity” in relation to a bacteriophagerefers to the type of host that said bacteriophage is able to infect.Specificity is usually mediated by the tail fibers of bacteriophages,which are involved in the interaction with receptors expressed on cells.A bacteriophage “specific” for P. aeruginosa more preferably designatesa bacteriophage which can infect one or several P. aeruginosa strainsand which cannot infect non-P. aeruginosa bacteria under physiologicalconditions.

As used herein, the term “polypeptide” refers to polypeptides of anysize, including small peptides of e.g., from 5 to 20 amino acids, longerpolypeptides, proteins or fragments thereof.

The term “PLE” or “Productive Lytic Effect” designates the ratio betweenburst size and productive lytic time of a given bacteriophage. Burstsize and productive lytic time are parameters defining phage-hostinteraction and correspond, respectively, to the mean yield ofbacteriophage particles produced by infection of one bacterium by onephage, and to the time taken by a free bacteriophage to lyse a bacterialcell.

In the context of the present specification, the term “isolatedbacteriophage” should be considered to mean material removed from itsoriginal environment in which it naturally occurs. In relation to abacteriophage, the term designates, particularly, a phage that is e.g.,cultivated, purified and/or cultured separately from the environment inwhich it is naturally located. In relation to a nucleic acid orpolypeptide, the term “isolated” designates e.g., a nucleic acidmolecule or polypeptide which is separated from at least some of thecomponents of its natural environment such as, e.g., a protein, lipid,and/or nucleic acid.

The term “pharmaceutically or veterinary acceptable” as used hereinrefers to any material (e.g., carrier, excipient or vehicle) that iscompatible for use in a mammalian subject. Such includes physiologicallyacceptable solutions or vehicles that are harmless or do not cause anysignificant specific or non-specific immune reaction to an organism ordo not abrogate the biological activity of the active compound. Forformulation of the composition into a liquid preparation, saline,sterile water, Ringer's solution, buffered physiological saline, albumininfusion solution, dextrose solution, maltodextrin solution, glycerol,ethanol, and mixtures thereof may be used as a pharmaceutically orveterinary acceptable excipient or carrier. If necessary, otherconventional additives such as thickeners, diluents, buffers,preservatives, surface active agents, antioxidants and bacteriostaticagents may be added. Further, diluents, dispersants, surfactants,binders and lubricants may be additionally added to the composition toprepare injectable formulations such as aqueous solutions, suspensions,and emulsions, oral formulations such as pills, capsules, granules, ortablets, or powdered formulations.

As used herein, “PFU” means plaque forming unit, as it is well definedin the art. Lytic bacteriophages lyse the host cell, causing a zone ofclearing (or plaque) on a culture plate. Theoretically, each plaque isformed by one phage and the number of plaques multiplied by the dilutionfactor is equal to the total number of phages in a test preparation.

The term “treatment” or “therapy” designates a curative or aprophylactic treatment of a disease. A curative treatment is defined asa treatment that results in a cure of a disease, or a treatment thatalleviates, reduces, stabilizes, or eliminates the symptoms of a diseaseor the suffering that it causes, directly or indirectly, or thatimproves a subject condition or reduces progression of a disease. Aprophylactic treatment comprises both a treatment resulting in theprevention of a disease and a treatment reducing and/or delaying theincidence of a disease or the risk of its occurrence.

The term “mammal” includes human subjects as well as non-human mammalssuch as pets (e.g., dogs, cats), horses, ruminants, sheep, goats, pigs,etc.

The term “biofilm” as used herein designates a heterogeneous bacterialformation growing on various surfaces; preferably a bacterial communitygrowing embedded in an exopolysaccharide matrix adhered onto solidbiological or non-biological surfaces.

The term “compromise” as used herein refers to any alteration of theintegrity. By compromising a bacterial biofilm, it is understood apenetration of the biofilm by bacteriophage, an infection ofbiofilm-associated bacteria and/or a lysis thereof and/or a partial oran entire clearing of the biofilm (i.e., by stopping colonization and/ordisrupting biofilms).

The term “sample”, as used herein, means any sample containing cells.Examples of such samples include fluids such as blood, plasma, saliva,or urine as well as biopsies, organs, tissues or cell samples. Thesample may be treated prior to its use.

As used herein, the term “subject” or “patient” refers to an animal,preferably to a mammal, even more preferably to a human, including adultand child. However, the term “subject” also encompasses non-humananimals, in particular mammals such as dogs, cats, horses, cows, pigs,sheep and non-human primates, among others.

The term “efficacy” of treatment or “response” to a bacteriophagetherapy as used herein refers to a treatment which results in a decreasein the number of P. aeruginosa strains in a subject after bacteriophagetreatment when compared to the number of P. aeruginosa strains beforetreatment. A “good responder” subject refers to a subject who shows orwill show a clinically significant recovery when treated with abacteriophage therapy.

The term “Cocktail” or composition of bacteriophages designates acombination of different types of bacteriophages. The bacteriophages ina cocktail/composition are preferably formulated together, i.e., in asame vessel or packaging, although they may be used as kits of partswherein the (or some of the) bacteriophages are formulated or packagedseparately and combined when used or administered.

DESCRIPTION OF EMBODIMENTS

The present invention is related to novel bacteriophage therapies. Moreparticularly, the present invention relates to novel bacteriophageshaving a high specificity against Pseudomonas aeruginosa strains, theirmanufacture, components thereof, compositions comprising the same andthe uses thereof in phage therapy.

Bacteriophages:

In a first aspect, the invention discloses the isolation andcharacterization of novel bacteriophages that are specific for P.aeruginosa strains and present, either alone or in combination(s),remarkable host range spectrum of lytic activity. These bacteriophageshave been selected from environmental samples, isolated, sequenced, andcharacterized. As indicated, the bacteriophages are, individually and incombination(s), active against P. aeruginosa strains. They areremarkably effective against pathogenic P. aeruginosa strains, such asantibiotic-resistant P. aeruginosa strains, such as an ESBL P.aeruginosa strain. Furthermore, bacteriophages of the invention have aremarkably productive lytic effect (“PLE”) below 20, more preferablybelow 15 and still more preferably between 0.3 and 15. Moreover, thebacteriophages of the invention are specific for P. aeruginosa strains,i.e., they do not cause lysis of non-P. aeruginosa bacteria. As will beillustrated further, the invention shows that these bacteriophages canbe combined and formulated in conditions suitable for use aspharmaceutical or veterinary agents to exhibit targeted and very potentantibacterial effect against a controlled spectrum of P. aeruginosastrains.

More specifically, the following bacteriophages have been selected andcharacterized. Their corresponding nucleic acid sequences are alsoindicated.

TABLE 1 SEQ ID number Bacteriophage SEQ ID NO: 1 BP1384 SEQ ID NO: 2BP1429 SEQ ID NO: 3 BP1430 SEQ ID NO: 4 BP1433 SEQ ID NO: 5 BP1450 SEQID NO: 6 BP1644 SEQ ID NO: 7 BP1647 SEQ ID NO: 8 BP1648 SEQ ID NO: 9BP1649 SEQ ID NO: 10 BP1650 SEQ ID NO: 11 BP1658 SEQ ID NO: 12 BP1661SEQ ID NO: 13 BP1662

The lytic profile of these bacteriophages has been determined on a broadnumber of P. aeruginosa strains. These bacteriophages have been selectedfor their potency and combination potential, as disclosed in thefollowing table. In this table, the lytic effect of the bacteriophageson reference and pathogen-resistant strains are presented, to confirmthe high lytic potential.

TABLE 2 Phage Bacteria 1384 1429 1430 1433 1450 1644 1647 1648 1649 16501658 1661 1662 LMG 24882 + + + + + + + + + + + + LMG 24883 ++/− + + + + + + + + + LMG 24886 + + + + +/− + +/− + LMG 24887 +/− +/−+/− + + +/− + + +/− +/− LMG 24891 + + + + + + + + + + + + LMG24892 + + + + + + + + +/− + + LMG 24893 + + + + + + + + + + + + + LMG24896 + + + + + + + + + + + + + LMG 24898 + + + + + + + + + + + + + LMG24901 +/− + +/− LMG 24903 +/− +/− + + +/− LMG 24904 +/− +/− +/− + +/−LMG 24905 +/− + + + + + LMG 24909 + +/− + + + + + + +/− + LMG 24913+/− + + +/− +/− LMG 24916 + + + + +

Further results on highly resistant strains from wound or burn arepresented below, further confirming the remarkable activity profile ofthe bacteriophages of the invention, and their complementarity.

TABLE 3 1384 1429 1430 1433 1450 1644 1647 1648 1649 1650 1658 1661 1662CAR* LMG + + + − + + + − + − + + − 1 25000 LMG − − − − + + − − + − + + −5 25122 LMG − + − − + + − − + − + + + 5 25140 LMG − − − + + − + − −− + + − 2 25133 LMG − − + − − − + − − − − + − 5 25165 LMG − − − − + + −− + − + + − 4 25146 *CAR: Class ATB Resistance

As can be seen from Tables 2 and 3, the phages have individually verystrong lytic power, and combinations (or cocktails) of thesebacteriophages may be produced that are able to kill all of the testedP. aeruginosa strains, thereby producing broad spectrum antibacterialcompositions.

As an illustration, a cocktail of all 13 phages of the invention is ableto effectively kill all bacteria listed in Table 2 and Table 3.

Moreover, the specificity of the bacteriophages has been tested on manynon-P. aeruginosa strains. More particularly, the Examples sectiondemonstrates that the bacteriophages of the invention have no lyticeffect on any bacteria selected from Escherichia coli, Acinetobacterbaumannii, Enterobacter aerogenes, Enterobacter asburiae, Enterobactercloacae, Klebsiella pneumonia, Porteus mirabilis, Staphylococcus aureus,Stenotrophomonas maltophilia and/or Serratia marcescens.

A particular object of the invention thus resides in a bacteriophagehaving lytic activity to a P. aeruginosa strain and having a genomecomprising a nucleotide sequence selected from any one of SEQ ID NOs: 1to 13 or a sequence having at least 97% identity thereto, preferably atleast 98% or 99% identity thereto.

The bacteriophages of the invention may be cultured, expanded, isolated,purified, and used in e.g., phage therapy of P. aeruginosa-mediateddisorders, as will be disclosed in more detail below. Furthermore,variants of these bacteriophages retaining a phenotypic (e.g.,specificity and lytic activity) of the bacteriophages can be producedand/or isolated by techniques known per se in the art.

The bacteriophages of the invention can be prepared by standard culture,isolation and purification methods. For example, P. aeruginosa producingbacteria are cultured, infected by a sample of a bacteriophage, and thentreated to remove bacterial cells and debris. The enriched bacteriophagesolution can be plated in a medium, for example agar medium, withembedded susceptible host strains of P. aeruginosa to obtain plaques.Then, single plaque can be picked out for subsequent bacteriophagepurification and amplification. One or more cycles of selectiveamplification of bacteriophages of the invention may be performed, forexample by mixing bacteriophages with the competent P. aeruginosa,followed by addition of a growth medium and incubation at selected testgrowing conditions. Following centrifugation, the cleared amplifiedsupernatant is filtered through filter and subjected to another cycle ofselective amplification or tested for presence of lytic activity.

The titer of phage in a suspension and the visualization of plaquemorphology of bacteriophages of the invention may then be assessed byknown methods, for example by plaque counting. Additionally, processingbacteriophages of the invention in various forms (liquid, lyophilized,etc.) for short-, long-, freeze- or any other kind of storage can becarried out by any suitable method as it is well-known in the art (seee.g., Clark, 1962).

The activity of the bacteriophages of the invention can be assessed bymethods well-known in the art, such as plaque assay also known as doubleagar method, based on the growing of bacteriophage with potential hostbacteria and followed by assessing their ability to kill the hostbacterial cell. In the plaque assay method, the bacteriophage induceslysis of target P. aeruginosa strains after a period of incubation insoft agar medium, resulting in zones of clearing on the plate known asplaques.

In a particular embodiment, the invention is related to BP1384bacteriophage, or any variant thereof. BP1384 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., P. aeruginosastrain PAO1. BP1384, or any variant thereof, is specific and has lyticactivity against LMG24882, LMG24883, LMG24886, LMG24891, LMG24892,LMG24893, LMG24896, LMG24898 and/or LMG24909 strains. BP1384 comprises agenome comprising a sequence as set forth in SEQ ID NO: 1 or having atleast 80% identity, more preferably at least 85% identity, and stillmore preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO: 1. It is also provided an isolated nucleic acid sequence fromBP1384 bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP1384 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP1384bacteriophage of the invention. BP1384 bacteriophage of the invention isalso characterized by a PLE below 20, more preferably below 15 and stillmore preferably of around 6.2.

In another particular embodiment, the invention is related to BP1429bacteriophage, or any variant thereof. BP1429 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., P. aeruginosastrain PAO1. BP1429, or any variant thereof, is specific and has lyticactivity against LMG24882, LMG24891, LMG24892, LMG24893, LMG24896,LMG24898 and/or LMG24916 strains. BP1429 comprises a genome comprising asequence as set forth in SEQ ID NO: 2 or having at least 80% identity,more preferably at least 85% identity, and still more preferably 90%,92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2. It is alsoprovided an isolated nucleic acid sequence from BP1429 bacteriophage, orvariant thereof. The invention also encompasses isolated polypeptidesencoded by BP1429 bacteriophage, or variant thereof, or encoded by anisolated nucleic acid sequence from BP1429 bacteriophage of theinvention. BP1429 bacteriophage of the invention is also characterizedby a PLE below 20, more preferably below 15 and still more preferably ofaround 0.70.

In still another aspect, the invention is related to BP1430bacteriophage, or any variant thereof. BP1430 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., P. aeruginosastrain PAO1. BP1430, or any variant thereof, is specific for and haslytic activity against LMG24882, LMG24883, LMG24891, LMG24892, LMG24893,LMG24896, LMG24898, LMG24901 and/or LMG24918 strains. BP1430 comprises agenome comprising a sequence as set forth in SEQ ID NO: 3 or having atleast 80% identity, more preferably at least 85% identity, and stillmore preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO: 3. It is also provided an isolated nucleic acid sequence fromB1430 bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP1430 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP1430bacteriophage of the invention. BP1430 bacteriophage of the invention isalso characterized by a PLE below 20, more preferably below 15 and stillmore preferably of around 3.

In another aspect, the invention is related to BP1433 bacteriophage, orany variant thereof. BP1433 bacteriophage, or any variant thereof, canbe produced or expanded in e.g., P. aeruginosa strain PAO1. BP1433, orany variant thereof, is specific and has lytic activity againstLMG24882, LMG24883, LMG24886, LMG24887, LMG24891, LMG24892, LMG24893,LMG24896, LMG24896, LMG24905, LMG24909 and/or LMG24916 strains. BP1433comprises a genome comprising a sequence as set forth in SEQ ID NO: 4 orhaving at least 80% identity, more preferably at least 85% identity, andstill more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identityto SEQ ID NO: 4. It is also provided an isolated nucleic acid sequencefrom BP1433 bacteriophage, or variant thereof. The invention alsoencompasses isolated polypeptides encoded by BP1433 bacteriophage, orvariant thereof, or encoded by an isolated nucleic acid sequence fromBP1433 bacteriophage of the invention. BP1433 bacteriophage of theinvention is also characterized by a PLE below 20, more preferably below15 and still more preferably of around 4.

In another particular embodiment, the invention is related to BP1450bacteriophage, or any variant thereof. BP1450 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., P. aeruginosastrain PAO1. BP1450, or any variant thereof, is specific for and haslytic activity against LMG24882, LMG24883, LMG24886, LMG24887, LMG24891,LMG24892, LMG24893, LMG24896, LMG24898, LMG24903, LMG24904, LMG24905,LMG24909 and/or LMG24913 strains. BP1450 comprises a genome comprising asequence as set forth in SEQ ID NO: 5 or having at least 80% identity,more preferably at least 85% identity, and still more preferably 90%,92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5. It is alsoprovided an isolated nucleic acid sequence from BP1450 bacteriophage, orvariant thereof. The invention also encompasses isolated polypeptidesencoded by BP1450 bacteriophage, or variant thereof, or encoded by anisolated nucleic acid sequence from BP1450 bacteriophage of theinvention. BP1450 bacteriophage of the invention is also characterizedby a PLE below 20, more preferably below 15 and still more preferably ofaround 2.

In still another aspect, the invention is related to BP1644bacteriophage, or any variant thereof. BP1644 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., P. aeruginosastrain PAO1. BP1644, or any variant thereof, is specific and has lyticactivity against LMG24882, LMG24883, LMG24886, LMG24891, LMG24892,LMG24893, LMG24896, LMG24898, LMG24905 and/or LMG24909 strains. BP1644comprises a genome comprising a sequence as set forth in SEQ ID NO: 6 orhaving at least 80% identity, more preferably at least 85% identity, andstill more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identityto SEQ ID NO: 6. It is also provided an isolated nucleic acid sequencefrom BP1644 bacteriophage, or variant thereof. The invention alsoencompasses isolated polypeptides encoded by BP1644 bacteriophage, orvariant thereof, or encoded by an isolated nucleic acid sequence fromBP1644 bacteriophage of the invention. BP1644 bacteriophage of theinvention is also characterized by a PLE below 20, more preferably below15 and still more preferably of around 1.5.

In another particular embodiment, the invention is related to BP1647bacteriophage, or any variant thereof. BP1647 bacteriophage, or anyvariant thereof, can be produced or expanded in, e.g., P. aeruginosastrain PAO1. BP1647, or any variant thereof, is specific for and haslytic activity against LMG24882, LMG24883, LMG24891, LMG24892, LMG24893,LMG24896, LMG24898, LMG24903 and/or LMG24916 strains. BP1647 comprises agenome comprising a sequence as set forth in SEQ ID NO: 7 or having atleast 80% identity, more preferably at least 85% identity, and stillmore preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO: 7. It is also provided an isolated nucleic acid sequence fromBP1647 bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP1647 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP1647bacteriophage of the invention. BP1647 bacteriophage of the invention isalso characterized by a PLE below 20, more preferably below 15 and stillmore preferably of around 0.4.

In another particular embodiment, the invention is related to BP1648bacteriophage, or any variant thereof. BP1648 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., P. aeruginosastrain PAO1. BP1648, or any variant thereof, is specific for and haslytic activity against LMG24882, LMG24883, LMG24891, LMG24893, LMG24896,LMG24898, and/or LMG24909 strains. BP1648 comprises a genome comprisinga sequence as set forth in SEQ ID NO: 8 or having at least 80% identity,more preferably at least 85% identity, and still more preferably 90%,92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 8. It is alsoprovided an isolated nucleic acid sequence from BP1648 bacteriophage, orvariant thereof. The invention also encompasses isolated polypeptidesencoded by BP1648 bacteriophage, or variant thereof, or encoded by anisolated nucleic acid sequence from BP1648 bacteriophage of theinvention. BP1648 bacteriophage of the invention is also characterizedby a PLE below 20, more preferably below 15 and still more preferably ofaround 2.

In another aspect, the invention is related to BP1649 bacteriophage, orany variant thereof. BP1649 bacteriophage, or any variant thereof, canbe produced or expanded in e.g., P. aeruginosa strain PAO1. BP1649, orany variant thereof, is specific for and has lytic activity againstLMG24882, LMG24883, LMG24886, LMG24887, LMG24891, LMG24892, LMG24893,LMG24896, LMG24898, LMG24905, LMG24909 and/or LMG24913 strains. BP1649comprises a genome comprising a sequence as set forth in SEQ ID NO: 9 orhaving at least 80% identity, more preferably at least 85% identity, andstill more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identityto SEQ ID NO: 9. It is also provided an isolated nucleic acid sequencefrom BP1649 bacteriophage, or variant thereof. The invention alsoencompasses isolated polypeptides encoded by BP1649 bacteriophage, orvariant thereof, or encoded by an isolated nucleic acid sequence fromBP1649 bacteriophage of the invention. BP1155 bacteriophage of theinvention is also characterized by a PLE below 20, more preferably below15 and still more preferably of around 3.5.

In another particular embodiment, the invention is related to BP1650bacteriophage, or any variant thereof. BP1650 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., P. aeruginosastrain PAO1. BP1650, or any variant thereof, is specific for and haslytic activity against LMG24882, LMG24893, LMG24896, LMG24898, LMG24905,and/or LMG24909 strains. BP1650 comprises a genome comprising a sequenceas set forth in SEQ ID NO: 10 or having at least 80% identity, morepreferably at least 85% identity, and still more preferably 90%, 92%,94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10. It is alsoprovided an isolated nucleic acid sequence from BP1650 bacteriophage, orvariant thereof. The invention also encompasses isolated polypeptidesencoded by BP1650 bacteriophage, or variant thereof, or encoded by anisolated nucleic acid sequence from BP1650 bacteriophage of theinvention. BP1650 bacteriophage of the invention is also characterizedby a PLE below 20, more preferably below 15 and still more preferably ofaround 14.

In still another aspect, the invention is related to BP1658bacteriophage, or any variant thereof. BP1658 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., P. aeruginosastrain PAO1. BP1658, or any variant thereof, is specific for and haslytic activity against LMG24882, LMG24887, LMG24891, LMG24893, LMG24896and/or LMG24898 strains. BP1658 comprises a genome comprising a sequenceas set forth in SEQ ID NO: 11 or having at least 80% identity, morepreferably at least 85% identity, and still more preferably 90%, 92%,94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11. It is alsoprovided an isolated nucleic acid sequence from BP1658 bacteriophage, orvariant thereof. The invention also encompasses isolated polypeptidesencoded by BP1658 bacteriophage, or variant thereof, or encoded by anisolated nucleic acid sequence from BP1658 bacteriophage of theinvention. BP1658 bacteriophage of the invention is also characterizedby a PLE below 20, more preferably below 15 and still more preferably ofaround 3.

In another aspect, the invention is related to BP1661 bacteriophage, orany variant thereof. BP1661 bacteriophage, or any variant thereof, canbe produced or expanded in e.g., P. aeruginosa strain PAO1. BP1661 orany variant thereof, is specific for and has lytic activity againstLMG24882, LMG24883, LMG24886, LMG24891, LMG24892, LMG24893, LMG24896,LMG24898, and/or LMG24909 strains. BP1661 comprises a genome comprisinga sequence as set forth in SEQ ID NO: 12 or having at least 80%identity, more preferably at least 85% identity, and still morepreferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ IDNO: 12. It is also provided an isolated nucleic acid sequence fromBP1661 bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP1661 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP1661bacteriophage of the invention. BP1661 bacteriophage of the invention isalso characterized by a PLE below 20, more preferably below 15 and stillmore preferably of around 4.

In still another aspect, the invention is related to BP1662bacteriophage, or any variant thereof. BP1662 bacteriophage, or anyvariant thereof, can be produced or expanded in e.g., P. aeruginosastrain PAO1. BP1662 or any variant thereof, is specific for and haslytic activity against LMG24883, LMG24891, LMG24892, LMG24893, LMG24896,LMG24898, and/or LMG24916 strains. BP1662 comprises a genome comprisinga sequence as set forth in SEQ ID NO: 13 or having at least 80%identity, more preferably at least 85% identity, and still morepreferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ IDNO: 13. It is also provided an isolated nucleic acid sequence fromBP1662 bacteriophage, or variant thereof. The invention also encompassesisolated polypeptides encoded by BP1662 bacteriophage, or variantthereof, or encoded by an isolated nucleic acid sequence from BP1662bacteriophage of the invention. BP1662 bacteriophage of the invention isalso characterized by a PLE below 20, more preferably below 15 and stillmore preferably of around 1.

Nucleic Acids and Polypeptides

The invention relates to a nucleic acid contained in a bacteriophage ofthe invention, or any fragment of such a nucleic acid. The term fragmentdesignates, more preferably, a fragment containing (or consisting of) anopen reading frame. The nucleic acid may be DNA or RNA, single- ordouble-stranded.

The nucleic acid can be isolated from the deposited bacteriophages, orproduced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning), enzymatic or chemical synthesis,or combinations thereof, according to general techniques known per se inthe art. Also included are homologous sequences and fragments thereofincluding, but not limited to, natural allelic variants and modifiednucleic acid sequences in which nucleotides have been inserted, deleted,substituted, and/or inverted.

In a particular embodiment, the invention relates to a nucleic acidcomprising a sequence selected from any one of SEQ ID NOs: 1-13, or asequence having at least 95%, 96%, 97%, 98%, 99% or more sequenceidentity to any one of SEQ ID NOs: 1-13.

In another particular embodiment, the invention relates to a nucleicacid comprising the sequence of a fragment of a sequence selected fromany one of SEQ ID NOs: 1-13, or a fragment of a sequence having at least95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ IDNOs: 1-13, said fragment comprising an open reading frame or aregulatory element such as a promoter.

The nucleic acid of the invention can be in free form, or cloned in avector.

In a further aspect, the invention also relates to an isolatedpolypeptide encoded by a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12 and SEQ ID NO: 13. The polypeptides may be produced bytechniques known per se in the art such as synthesis, recombinanttechnology, or combinations thereof. The polypeptides may be isolated orpurified, and used as antibacterial agents or as reagents for in vitroanalyses.

Compositions of the Invention

One aspect of the invention relates to compositions comprising at leastone bacteriophage as described above, more preferably at least 2 or moreand, optionally, a pharmaceutically or veterinary acceptable excipient.As described, the bacteriophages of the invention have very potent lyticactivity against P. aeruginosa strains. Combinations of thesebacteriophages may be produced to expand the host spectrum and producehighly effective antibacterial compositions.

More particularly, the invention relates to an antibacterial compositioncomprising at least two bacteriophages having lytic activity against aPseudomonas aeruginosa (P. aeruginosa) strain, said at least twobacteriophages being selected from the bacteriophages having a genomecomprising a nucleotide sequence of any one of SEQ ID NOs: 1 to 13 or asequence having at least 90% identity thereto, preferably at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In a preferred embodiment, the compositions of the invention comprise atleast three, even more preferably at least four distinct bacteriophagesselected from the bacteriophages having a genome comprising a nucleotidesequence of any one of SEQ ID NOs: 1 to 13 or a sequence having at least90% identity thereto, preferably at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity thereto. Compositions of the invention maycomprise at least 5, 6, 7, 8, 9, 10, 11, 11, 12 or all of the 13distinct types of bacteriophages as disclosed above.

One aspect of the invention is related to a composition at least onebacteriophage selected from BP1384, BP1429, BP1430, BP1433, BP1450,BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662, orvariants thereof.

The invention also concerns a composition comprising at least twodistinct bacteriophages selected from BP1384, BP1429, BP1430, BP1433,BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/orBP1662, or variants thereof.

In a particular embodiment, a composition of the invention comprisesBP1384 in combination with at least one further bacteriophage selectedfrom BP1429, BP1430, BP1433, BP1450, or BP1644.

In another particular embodiment, a composition of the inventioncomprises BP1384 in combination with at least one further bacteriophageselected from BP1450 and BP1647.

In another particular embodiment, a composition of the inventioncomprises BP1430 in combination with at least one further bacteriophageselected from BP1450, BP1644, BP1649 and BP1661.

In another particular embodiment, the composition comprises BP1433 incombination with at least one further bacteriophage selected fromBP1450, BP1647, BP1648, BP1650 and BP1658.

In another preferred embodiment, the composition comprises BP1384 incombination with at least one further bacteriophage selected fromBP1429, BP1647, BP1649 and BP1662.

The invention also relates to a composition comprising a combination ofall of the bacteriophages BP1384, BP1429, BP1430, BP1433, BP1450,BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662, orvariants thereof.

Specific examples of compositions of the invention comprise:

-   -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 1 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 4 or a sequence having at        least 90% identity thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 1 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 5 or a sequence having at        least 90% identity thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 3 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 9 or a sequence having at        least 90% identity thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 3 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 4 or a sequence having at        least 90% identity thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 3 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 10 or a sequence having at        least 90% identity thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 1 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 3 or a sequence having at        least 90% identity thereto, and a bacteriophage having a genome        comprising a nucleotide sequence of SEQ ID NO: 4 or a sequence        having at least 90% identity thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 1 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 3 or a sequence having at        least 90% identity thereto, and a bacteriophage having a genome        comprising a nucleotide sequence of SEQ ID NO: 5 or a sequence        having at least 90% identity thereto; or    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 1 or a sequence having at least 90% identity        thereto, and a bacteriophage having a genome comprising a        nucleotide sequence of SEQ ID NO: 3 or a sequence having at        least 90% identity thereto, and a bacteriophage having a genome        comprising a nucleotide sequence of SEQ ID NO: 9 or a sequence        having at least 90% identity thereto.

A specific embodiment of the invention relates to a compositioncomprising:

-   -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 1 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 2 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 3 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 4 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 5 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 6 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 7 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 8 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 9 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 10 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 11 or a sequence having at least 90% identity        thereto;    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 12 or a sequence having at least 90% identity        thereto; and    -   a bacteriophage having a genome comprising a nucleotide sequence        of SEQ ID NO: 13 or a sequence having at least 90% identity        thereto.

The compositions of the invention may further comprise additionalantibacterial agents, particularly other bacteriophages having distincthost specificity.

Preferred compositions of the invention are lytic againstantibiotic-resistant P. aeruginosa strains.

Further preferred compositions of the invention are lytic against morethat 90% of all bacterial strains of the LMG collection, obtained fromthe well-known BCCM/LMG Bacteria Collection. This collection isaccessible via Worldwide Website:cabri.org/CABRI/srs-doc/bccm_lmg.info.html.

The antibacterial compositions of the invention may be in various forms,such as liquid, semi-liquid, solid or lyophilized formulations.

The compositions of the invention may comprise any effective amount ofthe selected bacteriophage(s). Preferably, they comprise between 10^(e4)and 10^(e12) PFU of each of said bacteriophages, preferably between10^(e5) and 10^(e10) PFU. The relative amounts of each type ofbacteriophage in a composition of the invention may be adjusted by askilled artisan. Typically, when the antibacterial composition comprisesseveral (n) distinct bacteriophages as defined above, the total relativeamount % A of each bacteriophage in the composition is more preferably %A=(100/n_(i))×V, wherein n_(i) represents the number of distinct typesof bacteriophages and V is a variability factor comprised between 0.2and 5. Most preferably, V is comprised between 0.3 and 3, even morepreferably between 0.5 and 2, generally between 0.8 and 1.5. In apreferred typical embodiment, each type of bacteriophage is present in acomposition of the invention in approximately equal relative amounts.

The compositions of the invention preferably comprise a suitable diluentor carrier, such as a pharmaceutically or veterinary acceptableexcipient or carrier. Compositions according to the present inventionmay include any excipient or carrier, such as thickeners, diluents,buffers, preservatives, surface active agents and the like, in additionto the bacteriophage(s) of choice. Such includes physiologicallyacceptable solutions or vehicles that are harmless or do not cause anysignificant specific or non-specific immune reaction to an organism ordo not abrogate the biological activity of the bacteriophage. For liquidformulation, saline, sterile water, Ringer's solution, bufferedphysiological saline, albumin infusion solution, dextrose solution,maltodextrin solution, glycerol, ethanol, and mixtures thereof may beused as a pharmaceutically or veterinary acceptable excipient orcarrier. If appropriate, other conventional additives such asthickeners, diluents, buffers, preservatives, surface active agents,antioxidants and bacteriostatic agents may be added. Further, diluents,dispersants, surfactants, binders and lubricants may be additionallyadded to the composition to prepare injectable formulations such asaqueous solutions, suspensions, and emulsions, oral formulations such aspills, capsules, granules, or tablets, or powdered formulations.Formulations for topical administration may include, band aids,dressings, patches, films, ointments, lotions, creams, gels, drops,suppositories, sprays, tampons, sanitary towels, liquids and powders.Formulations for decontamination or for medical use may also includeaerosols or sprays.

The compositions of the invention may be used in the medical field,including the human or veterinary medical areas, for e.g. the treatmentof an infection in a mammal or for improving a subject's condition. Thecompositions may be used to kill P. aeruginosa bacteria in an organism,for treating an infection. The composition may also be used forimproving the condition of a mammal by modifying the microbial flora insaid mammal. In particular, the compositions of the invention canspecifically remove P. aeruginosa strains on the skin or mucousmembranes of a mammal, thus modifying its microbial flora and restoringa proper balance.

In a particular embodiment, the invention also relates to a method fortreating an infection in a mammal comprising the administration to saidmammal of a composition or bacteriophage or nucleic acid or polypeptideas defined above. In a particular embodiment the method comprisesadministering at least one, preferably at least two, even morepreferably at least three bacteriophages selected from BP1384, BP1429,BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649, BP1650, BP1658,BP1661 and/or BP1662, or variants thereof.

The invention also relates to the use of a composition, bacteriophage,nucleic acid or polypeptide as described for the manufacture of amedicament for treating an infection in a mammal, or for restoringmicrobial flora in said mammal.

The compositions or agents of the invention may be administered by anyconvenient route, including intravenous, oral, transdermal,subcutaneous, mucosal, intramuscular, intrapulmonary, intranasal,parenteral, rectal, vaginal and topical. In a preferred embodiment, thebacteriophages or compositions are administered by topical route, e.g.,by application on the skin of a subject. The compositions may beadministered directly or indirectly, e.g., via a support. In thisregard, the compositions may, for example, be applied or sprayed to theafflicted area. Compositions of the invention can also be administeredby oral or parenteral routes. The dosage suitable for applying,spraying, or administering the compositions of the present invention canbe adjusted by the skilled person depending on a variety of factorsincluding formulation, mode of administration, age, weight, sex,condition, diet of the mammal being treated at the time ofadministration, route of administration, and reaction sensitivity. Aphysician having ordinary skill in the art can readily determine andprescribe the effective amount of the composition required.

The dosing can also be adjusted by the skilled person so that a lyticactivity against antibiotic-resistant P. aeruginosa strains is obtained.An efficient dose to obtain a lytic activity in vivo typically includesa concentration of at least 10^(e2) PFU/ml, preferably from about10^(e2) to 10^(e12) PFU/ml, depending on the administration route.Administration may be performed only once or, if needed, repeated.

The compositions of the invention may be administered to treat P.aeruginosa infections, typically of the respiratory tract, urinarytract, burns, wounds, ear, skin, or soft tissues, or gastrointestinal orpost-surgical infections.

As shown in the Examples section, the bacteriophages and compositions ofthe invention are able to selectively kill P. aeruginosa bacteria invitro or in vivo. The compositions can destroy mixtures of different Paeruginosa bacteria, even in vivo, even at low dosage. Furthermore, thecompositions of the invention are effective at killing bacteria embeddedin biofilms, which is particularly important for pathogenic bacteria.Also, the compositions and bacteriophages of the invention are strictlyunable to affect mammalian cells, and are therefore specific and devoidof side effects in vivo.

The invention also relates to the use of a composition, bacteriophage,nucleic acid or polypeptide of the invention for decontaminating amaterial. Due to their potent antibacterial effect, and to their abilityto even compromise the integrity of a bacterial biofilm, thecompositions of the invention can be used as decontaminating agent, toeliminate or at least cause a reduction in bacterial numbers on amaterial. Such methods may be applied for the treatment of a variety ofbiological or non-biological surfaces in both medical and non-medicalcontexts, including solid materials or devices such as, for example,contact lenses, surfaces of devices to be implanted into the body,pipes, ducts, laboratory vessels, textiles, etc.

Diagnostic/Predictive Tests of the Invention:

The invention also concerns a method for predicting or determining theefficacy of a bacteriophage therapy in a subject, wherein the methodcomprises a step of determining a lytic activity of one or morebacteriophages selected from BP1384, BP1429, BP1430, BP1433, BP1450,BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662 toa P. aeruginosa strain from a sample from said subject, such lyticactivity being indicative of an efficient treatment. In a preferredaspect, the method further optionally comprises a step of treating saidsubject by one or more bacteriophages having a lytic activity to a P.aeruginosa strain from a sample of said subject.

In another aspect, the invention provides a method for selecting asubject or determining whether a subject is susceptible to benefit froma bacteriophage therapy, wherein the method comprises the step ofdetermining a lytic activity of one or more bacteriophages selected fromBP1384, BP1429, BP1430, BP1433, BP1450, BP1644, BP1647, BP1648, BP1649,BP1650, BP1658, BP1661 and/or BP1662 to a P. aeruginosa strain from asample of said subject, a lytic activity of one or more bacteriophagesof the invention to at least one P. aeruginosa strain indicating aresponder subject.

Another object of the invention relates to a method for predicting theresponse of a subject to a bacteriophage therapy, wherein the methodcomprises the step of determining a lytic activity of one or morebacteriophage selected from BP1384, BP1429, BP1430, BP1433, BP1450,BP1644, BP1647, BP1648, BP1649, BP1650, BP1658, BP1661 and/or BP1662 toa P. aeruginosa strain from a sample of said subject, a lytic activityof one or more bacteriophage of the invention to at least one P.aeruginosa strain being indicative of a good response to said therapy.

Further aspects and advantages of the invention will be disclosed in thefollowing experimental section, which is illustrative only.

EXAMPLES

Materials and Methods

Phage Isolation and Preparation

MDR P. aeruginosa bacteria were used for isolating and enriching eachvirulent bacteriophage from environmental water. Environmental samplesand overnight culture of bacteria in Luria Bertani (LB) were mixed andincubated at 37° C. for 24 h with shaking to enrich specificbacteriophages. At the end of incubation, drops of chloroform were addedto the culture. The culture was spun down at 11,000 g for 5 minutes toremove bacterial cells and debris. The supernatant was subjected to 0.2μm filter to remove the residual bacterial cells. The enriched phagesolution was plated on LB agar medium with P. aeruginosa embedded.Plaques formed on the plates after 24 h incubation at 37° C. Singleplaque was picked out for subsequent phage purification andamplification. The phage was then stored at 4° C. in a suspension in LBbroth or physiological saline.

The titer of phage in a suspension was estimated by plaque counting(Postic, 1961). 10-fold dilutions of a suspension were delivered on adried lawn of the propagating strain. The plates were read afterovernight incubation. The plaque-counting method also permittedvisualization of plaque morphology.

Host Range Determination

The host ranges of bacteriophages were determined among a collection of20 P. aeruginosa from the LMG collection. 10⁹ bacterial cells were mixedwith melted agar and this mixture was poured on solid agar to makedouble layer agar plates. After solidification, isolated bacteriophagestock solutions were spotted on each plate with different bacteriumstrain. After allowing 20 min for the spots to be absorbed, the plateswere inverted and incubated for 24 h at 37° C. before the degree oflysis was recorded (Postic, 1961; Yang, 2010).

Electron Microscopy

Electron micrographs of each phage were taken with a transmissionelectron microscope.

Sequencing, Analysis and Annotation of Phage Genomes

To isolate phage DNA, phages were propagated as described above. PhageDNA was isolated by extraction with phenol:chloroform:isoamyl alcohol(25:24:1, V/V), ethanol precipitation and resolution in water. Wholegenome sequencing was done and the BLAST algorithm was used to determinethe similarity to described genes in the National Center forBiotechnology Information (NCBI) database. The genomes were scanned forpotential open reading frames (ORFs).

Example 1: Bacteriophage-Host Characteristics and Kinetics

One-step growth experiments were carried out according to the previousdescriptions to determine first the productive lytic time, adsorptionrate then the phage burst size. To determine the adsorption rate sampleswere taken at different time intervals to analyze the free phageparticles in the solutions. For productive time and phage burst sizedetermination, P. aeruginosa bacteria were mixed with phage solutionsand phages were allowed to adsorb for 15 min. The mixture was subjectedto centrifugation immediately at 5000 rpm for 10 min to remove freephage particles. The pellet was resuspended in 5 fresh LB medium and theculture was continuously incubated at 37° C. Samples were taken at 3 minintervals and phage titre was determined. These results permitted tocalculate the number of phages produced per bacteria (burst size), theproductive time and the productive lytic effect (PLE), as shown in Table5 below.

TABLE 4 PLE Productive BURST SIZE (PFU per lytic time Adsorption rate(PFU per bacterium Phage (min) (ml−1min−1) bacterium) per min) 1384 808.64E−09 499 6.24 1429 70 9.16E−09 49 0.70 1430 60 1.66E−08 166 2.761433 100 2.72E−09 399 3.99 1450 100 1.07E−08 199 1.99 1644 70 8.35E−0999 1.41 1647 90 2.34E−08 32 0.36 1648 100 2.70E−09 199 1.99 1649 1001.10E−08 332 3.32 1650 70 3.47E−09 999 14.27 1658 90 1.61E−08 249 2.771661 90 7.11E−09 332 3.69 1662 90 9.16E−09 99 1.10

These results show that all phages have potent viral production capacityand absorption rates. Most phages have a PLE below 7, which demonstratesa remarkable profile. Phages 1429 and 1647 are particularly effective inthis regard. In addition, the different PLE and adsorption times permitto create cocktails with selected variability.

Example 2: Preparation of Cocktail Compositions

The following cocktail compositions are constituted, each comprisingbetween 10-9 and 10-11 pfu of each bacteriophage:

TABLE 5 Cocktail Phages I P1384 + P1433 II P1384 + P1450 III P1430 +P1649 IV P1430 + P1433 V P1430 + P1650 VI P1384 + P1430 + P1433 VIIP1384 + P1430 + P1450 VIII P1384 + P1430 + P1649

The following additional two cocktail compositions comprising all of thevarious phages are constituted, covering the most important diversity ofP. aeruginosa species:

Cocktail composition A: Phage 1384 1429 1430 1433 1450 1644 1647 1648titer 4.00E+10 1.23E+09 5.45E+08 8.33E+10 8.91E+10 9.09E+08 2.00E+093.09E+09 Phage 1649 1650 1658 1661 1662 Titre 9.00E+09 9.45E+08 1.91E+091.14E+09 3.55E+08

Cocktail composition B: phage 1384 1429 1430 1433 1450 1644 1647 1648Titre 1.60E+11 2.00E+11 2.00E+11 1.20E+11 8.00E+10 1.00E+11 1.00E+081.00E+09 phage 1649 1650 1658 1661 1662 Titre 1.00E+11 2.20E+11 8.00E+101.00E+11 6.00E+07

Example 3: Sensitivity of Bacteria to Bacteriophage Cocktails of theInvention

Various strains of bacteria were tested with the bacteriophage cocktailsof the invention at 2.10⁹ bacteriophages/ml. Different bacterialconcentrations were plated on the bacteriophage cocktail at 2.10⁹bacteriophages/ml and incubated 24 h at 37° C.

Cocktails are tested on the 22 distinct P. aeruginosa bacteria listed inTables 2 and 3. The % of bacteria species sensitive to the cocktails arelisted in Table 6 below:

TABLE 6 Cocktail % Killed P. aeruginosa species I 73% II 82% III 91% IV86% V 77% VI 86% VII 95% VIII 95% A 100%  B 100% 

Bacteria were enumerated and used to the calculation of resistance rate(number of bacteria after incubation/number of bacteria plated).Resistance rates with a cocktail comprising the 13 different types ofbacteriophages are shown in Table 7 below:

TABLE 7 Bacteria Rate (bacteria/ml) LMG 24891 1.00E−05 LMG 249455.80E−06 LMG 24970 1.00E−05 LMG 25082 4.60E−06 LMG 25131 9.00E−06 LMG25194 9.00E−06

All tested bacteria are sensitive to compositions of the invention.

Example 4: Cocktail Specificity

The cocktail specificity was confirmed by testing on ten bacteriaspecies, including Escherichia coli, Acinetobacter baumannii,Enterobacter aerogenes C, Enterobacter asburiae, Enterobacter cloacae,Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus,Stenotrophomonasmaltophilia, and Serratia marcescens.

Table 8 summarizes lytic activity observed for each bacteriophage usedindependently or in combination as a cocktail of 13 bacteriophages.

Cocktail Phage PYO 1384 1429 1430 1433 1450 1644 1647 1648 1649 16501658 1661 1662 13 phages Pseudomonas aeruginosa SH 85 +/− +/− +/− +/−+/− + +/− +/− +/− + + SH 224 + + +/− + + E. coli SH 213 − SH 141 −Acinebacter baumanii SH 32 − SH 34 − Enterobacter aerogenes C SH 97 − SH98 − Enterobacter asburiae SH 74 − Enterobacter cloacae SH 111 − SH 121− Enterobacter amnigenus SH 26 − Klebsiella pneumoniae SH 89 − SH 283 −Proteus mirabilis SH 82 − S. aureus (meti R) SH 14 − SH 129 −Stenotrophomonas maltophila SH 286 − SH 290 − Serratia marcescens SH 314−

The above table clearly show that no lytic activity on bacteria otherthan P. aeruginosa strain occurred. The bacteriophages and cocktail ofthe invention are therefore highly specific for P. aeruginosa strains.

Example 5: Efficiency of Bacteriophages on P. aeruginosa Strain In Vitro

Several strains of the LMG collection were chosen to represent thegenetic diversity of P. aeruginosa and various forms of antibioticresistance. Strains were either sensitive or resistant to one or severalantibiotics, as described in Table 9. They were grown individually or incombination with 2 to 8 strains. The bacteriophage cocktail was added atan MOI of 1 to 10^(e-6), i.e. at a dilution ratio (bacteria/phage) of 1to 1 million.

TABLE 9 Information about the bacterial strains Sero- Class ATB LMG No.Country Year Source type resistance LMG 24891 France 1882-1918 Surgical11 1 bandage LMG 24893 Greece 1994 Sputum 11 2 LMG 24909 Colombia 2003Peritoneal 12 0 fluid LMG 24988 Turkey 1997 Burn  8 3 LMG 24992 UK 2003CF-patient NT 4 LMG 25041 The 1993 Wound NT 2 Philippines LMG 25049France 1882-1918 Wound  6 1 LMG 25140 Panama 2006 Wound 11 5

The results are presented in FIG. 1 and in Table 10.

TABLE 10 Efficiency of bacteriophage cocktail obtained in vitro on P.aeruginosa mixture at 2.10^(e7) cfu/ml and at various dilutions Mix of:MOI 1 MOI 0.1 MOI 0.01 MOI 0.001 MOI 0.0001 MOI 0.000001 1 bacterium ++++ ++ ++ ++ ++ 2 bacteria ++ ++ ++ ++ ++ ++ 3 bacteria ++ ++ + + + + 4bacteria ++ ++ + +/− +/− +/− 5 bacteria ++ + + +/− +/− 6 bacteria ++ + ++/− +/− 7 bacteria ++ + + +/− +/− 8 bacteria ++ + +/− +/−

The compositions of the invention are able to kill a mixture of 8distinct strains of P. aeruginosa bacteria together. The cocktailremains efficient against 8 strains at a dilution of 1/1000.

Example 6: Efficiency of Bacteriophages on P. aeruginosa Strain In Vivo

An isolated Is580 strain, collected from a burned patient in 1997, wasused for the following experiments.

Is580 strain is resistant to ampicillin, AMC, PIP, CEF, CXM, axetil CXM,FOX, CPD, CTX, CAZ, GEN, TOB, OFX, NIT, and SXT.

SKH1 mouse (or hairless mouse) was used as mouse model of P. aeruginosainfection.

Modus Operandi: (See Table 11 Below):

-   -   Mice were immunodepressed by 3 IP injections of 1.5 mg of        cyclophosphamide (Cy), every 2 days from Day −3 before        infection.    -   Mice were burned on skin by 2 μl of liquid yperite at 30 mg/kg.    -   Infection two days after the burn by subcutaneous injection of a        bacterium suspension in burned site.

TABLE 11 Day −3 −2 −1 0 1 1.5 mg Burn 1.5 mg Infection 1.5 mg Cy Cy CyInjection IP Yperite IP SC 10⁷ cfu IP route PHAGE SC injection ofcocktail (100 μl, i.e. 10⁸ PFU) 6 h post-infection

Cocktail compositions were prepared according to Example 1 andcompresses soaked of bacteriophages cocktail at 10^(e7) phages/ml wereapplied at Day 0.

Various concentrations of P. aeruginosa strains were tested with 100 μlof bacteriophage cocktail. As shown on FIG. 2, all P. aeruginosa strainswere killed 6 h post-treatment.

Upon administration of Is580 P. aeruginosa strain by subcutaneousinjection to SKH1 mice, only 35% of mice survived in the absence offurther treatment. In the mice treated by injection of a bacteriophagecocktail as presented in Table 10 above, a remarkable survival rate wasobserved (see FIG. 3): 100% survival for SKH1 mice treatedsubcutaneously, 16 days after infection. By comparison, a 50% survivalwas observed for SKH1 mice treated by antibiotic 2 days after infection.

Accordingly, the compositions of the invention can treat an infection invivo and can induce a 100% survival rate in infected mice.

REFERENCES

-   Clark W A, 1962, Appl Microbiol. Comparison of several methods for    preserving bacteriophages. 1962 September; 10:466-71.-   Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B, Delattre A S,    Lavigne R, 2012, Learning from bacteriophages—advantages and    limitations of phage and phage-encoded protein applications. Curr.    Protein Pept. Sci. 13(8):699-722.-   Fordos J. 1859. Receuil des travaux de la Societé d'Emulation pour    les Sciences Pharmaceutiques, vol 3 Societé d'Emulation pour les    Sciences Pharmaceutiques, Paris, France.-   Freeman L. 1916. Chronic general infection with the Bacillus    pyocyaneus. Ann. Surg. 64:195-202.-   Gang R K, Bang R L, Sanyal S C, Mokaddas E, Lari A R. 1999.    Pseudomonas aeruginosa septicaemia in burns. Burns 25:611-616.-   Jones A M, et al. 2010. Clinical outcome for cystic fibrosis    patients infected with transmissible Pseudomonas aeruginosa: an    8-year prospective study. Chest 137:1405-1409.-   Kang C I, et al. 2005. Bloodstream infections caused by    antibiotic-resistant gram-negative bacilli: risk factors for    mortality and impact of inappropriate initial antimicrobial therapy    on outcome. Antimicrob. Agents Chemother. 49:760-766.-   Micek S T, et al. 2005. Pseudomonas aeruginosa bloodstream    infection: importance of appropriate initial antimicrobial    treatment. Antimicrob. Agents Chemother. 49:1306-1311.-   Strateva T. and Yordanov D. 2009. Pseudomonas aeruginosa—a    phenomenon of bacterial resistance. Journal of Medical Microbiology    58, 1133-1148.-   Weinbauer M G. Ecology of prokaryotic viruses. FEMS Microbiol Rev    2004; 28:127-81.-   Williams E P, Cameron K. 1894. Infection by the Bacillus pyocyaneus    a cause of infantile mortality. Public Health Pap. Rep. 20:355-360.

We claim:
 1. A method of treating a mammal comprising the administrationto said mammal of at least one bacteriophage having lytic activity to aPseudomonas aeruginosa (P. aeruginosa) strain, wherein said at least onebacteriophage has a genome comprising the nucleotide sequence of SEQ IDNO: 5 or a sequence having at least 98% identity thereto.
 2. The methodof claim 1, wherein said at least one bacteriophage has a genomecomprising the nucleotide sequence of SEQ ID NO: 5 or a sequence havingat least 99% identity thereto.
 3. The method of claim 1, wherein said atleast one bacteriophage has a genome comprising the nucleotide sequenceof SEQ ID NO:
 5. 4. The method of claim 1, further comprising thecombined or separate administration to said mammal of at least oneadditional bacteriophage.
 5. The method of claim 4, wherein said atleast one additional bacteriophage has lytic activity to a P. aeruginosastrain and has a genome comprising a nucleotide sequence of any one ofSEQ ID NOs: 1 to 4 and 6 to 13 or a sequence having at least 97%identity thereto.
 6. The method of claim 1, wherein the at least onebacteriophage is formulated in a composition with a pharmaceuticallyacceptable excipient or carrier.
 7. The method of claim 6, wherein thecomposition is a liquid, semi-liquid, solid or lyophilized formulation.8. The method of claim 1, which comprises the administration of between10^(e4) and 10^(e12) PFU of said at least one bacteriophage.
 9. Themethod of claim 1, wherein the mammal is a human.
 10. The method ofclaim 1, wherein the mammal has a bacterial infection.